Streptococcus pyogenes, also known as xcex2-hemolytic group A streptococci (GAS) is a pathogen of humans which can cause mild infections such as pharyngitis and impetigo. Post infection autoimmune complications can occur, namely rheumatic fever and acute glomerulonephritis. GAS also causes severe acute diseases such as scarlet fever and streptococcal toxic shock syndrome (STSS). Severe GAS infections were a large problem in the U.S. and throughout the world at the beginning of this century. In the mid-forties, the number of cases and their severity decreased steadily for yet not completely understood reasons. However, more recently, a resurgence of serious diseases caused by GAS has been seen such that there may be 10-20,000 cases of STSS each year in the United States. As many as 50 to 60% of these patients will have necrotizing fascitis and myositis; 30 to 60% will die and as many as one-half of the survivors will have limbs amputated.
In 1986 and 1987 two reports described an outbreak of severe GAS infections localized in the Rocky Mountain area. These reports have been followed in the past few years by several others describing a disease with analogous clinical presentation. The symptoms described for this disease were very similar to those described for toxic shock syndrome (TSS), and in 1992 a committee of scientists gave to this clinical presentation the formal name of STSS, and set the criteria for its diagnosis. STSS is defined by the presence of the following:
1. hypotension and shock;
2. isolation of group A streptococci;
3. two or more of the following symptoms: fever 38.5xc2x0 C. or higher, scarlet fever rash, vomiting and diarrhea, liver and renal dysfunction, adult respiratory distress syndrome, diffuse intravascular coagulation, necrotizing fascitis and/or myositis, bacteremia.
Streptococcal isolates from STSS patients are predominantly of M type 1 and 3, with M18 and nontypable organisms making up most of the reminder. The majority of M1, 3, 18, and nontypable organisms associated with STSS make pyrogenic exotoxin A (SPE-A, scarlet fever toxin A). In contrast, only 15% of general streptococcal isolates produce this toxin. Moreover, administration of SPE-A to a rabbit animal model and in two accidental human inoculations can reproduce the symptoms of STSS.
SPE-A is a single peptide of molecular weight equal to 25,787 daltons, whose coding sequence is carried on the temperate bacteriophage T12. speA, the gene for SPE-A, has been successfully cloned and expressed in Escherichia coli. SPE-A is a member of a large family of exotoxins produced by streptococci and staphylococci, referred to as pyrogenic toxins based upon their ability to induce fever and enhance host susceptibility up to 100,000 fold to endotoxin.
Recently these toxins have been referred to as superantigens because of their ability to induce massive proliferation of T lymphocytes, regardless of their antigenic specificity, and in a fashion dependent on the composition of the variable part of the xcex2 chain of the T cell receptor. These toxins also stimulate massive release of IFN-xcex3, IL-1, TNF-xcex1 and TNF-xcex2. Other members of this family are streptococcal pyrogenic exotoxins type B and C, staphylococcal toxic shock syndrome toxin 1, staphylococcal enteroxtoxins A, B, Cn, D, E. G and H, and non-group A streptococcal pyrogenic exotoxins. These toxins have similar biochemical properties, biological activities and various degrees of sequence similarity.
The most severe manifestations of STSS are hypotension and shock, that lead to death. It is generally believed that leakage of fluid from the intravascular to the interstitial space is the final cause of hypotension, supported by the observation that fluid replacement therapy is successful in preventing shock in the rabbit model of STSS described above. It has been hypothesized that SPE-A may act in several ways on the host to induce this pathology. Certain single amino acid substitutions in central regions of the SPE-A molecule have been shown to affect the mitogenic activity of and binding to a HLA class II molecules by SPE-A (Hartwig et al. International Immunology 5:5, 869-875 (1993)).
SPE-A has been shown to block liver clearance of endotoxin of endogenous flora""s origin, by comprising the activity of liver Kuppfer cells. This appears to cause a significant increase in circulating endotoxin, that through binding to lipopolysaccharide binding protein (LBP) and CD14 signaling leads to macrophage activation with subsequent release of TNF-xcex1 and other cytokines. Support for the role of endotoxin in the disease is given by the finding that the lethal effects of SPE-A can be at least partially neutralized by the administration to animals of polymyxin B or by the use of pathogen free rabbits.
Another modality of induction of shock could be the direct activity of the toxin on capillary endothelial cells. This hypothesis stems from the finding that the staphylococcal pyrogenic toxin TSST-1 binds directly to the human umbilical cord vein cells and is cytotoxic to isolated porcine aortic endothelial cells.
Another of the toxin""s modality of action includes its superantigenicity, in which the toxin interacts with and activates up to 50% of the host T lymphocytes. This massive T cell stimulation results in an abnormally high level of circulating cytokines TNF-xcex2 and IFN-xcex3 which have direct effects on macrophages to induce release of TNF-xcex1 and IL-1. These cytokines may also be induced directly from macrophages by SPE-A through MHC class II binding and signalling in the absence of T cells. The elevated levels of TNF-xcex1 and -xcex2 cause several effects typically found in Gram negative induced shock, among which is damage to endothelial cells and capillary leak. However, the administration to SPE-A treated rabbits of cyclosporin A, which blocks upregulation of IL-2 and T cell proliferation, did not protect the animals from shock, suggesting that additional mechanisms may be more important in causing capillary leak.
Thus, there is a need to localize sites on the SPE-A molecule responsible for different biological activities. There is a need to develop variants of SPE-A that have changes in biological activities such as toxicity and mitogenicity. There is a need to develop compositions useful in vaccines to prevent or ameliorate streptococcal toxic shock syndrome. There is also a need to develop therapeutic agents useful in the treatment of streptococcal toxic shock syndrome and other diseases.
This invention includes mutant SPE-A toxins and fragments thereof, vaccines and pharmaceutical compositions and methods of using vaccines and pharmaceutical compositions.
Mutant SPE-A toxins have at least one amino acid change and are substantially nonlethal as compared with a protein substantially corresponding to a wild type SPE-A toxin. For vaccine compositions, mutant toxins also stimulate a protective immune response to at least one biological activity of a wild type SPE-A toxin. Mutant toxins for vaccine compositions are optionally further selected to have a decrease in enhancement of endotoxin shock and a decrease in T cell mitogenicity when compared to the wild type SPE-A. An especially preferred mutant for vaccine compositions is one that has a change at an amino acid equivalent to amino acid 20 of a wild type SPE-A toxin. For pharmaceutical compositions, it is preferred that a mutant toxin is substantially nonlethal while maintaining T cell mitogenicity comparable to a wild type SPE-A toxin.
The invention also includes fragments of a wild type speA toxin and mutants of speA toxins. Fragments and peptides derived from wild type SPE-A are mutant SPE-A toxins. Fragments can include different domains or regions of the molecule joined together. A fragment is substantially nonlethal when compared to a wild type SPE-A toxin. For mutant toxins, a fragment has at least one amino acid change compared to a wild type SPE-A amino acid sequence. Fragments are also useful in vaccine and pharmaceutical compositions.
The invention also includes expression cassettes, vectors and transformed cells. An expression cassette comprises a DNA sequence encoding a mutant SPE-A toxin or fragment thereof operably linked to a promoter functional in a host cell. DNA cassettes are preferably inserted into a vector. Vectors include plasmids or viruses. Vectors are useful to provide template DNA to generate DNA encoding a mutant SPE-A toxin. DNA cassettes and vectors are also useful in vaccine compositions. Nucleic acids encoding a mutant SPE-A toxin or fragment thereof can be delivered directly for expression in mammalian cells. The promoter is preferably a promoter functional in a mammalian cell. Nucleic acids delivered directly to cells can provide for expression of the mutant SPE-A toxin in an individual so that a protective immune response can be generated to at least one biological activity of a wild type SPE-A toxin.
Additional vaccine composition include stably transformed cells or viral vectors including an expression cassette encoding a mutant SPE-A toxin or fragment thereof. Viral vectors such as vaccinia can be used to immunize humans to generate a protective immune response against at least one biological activity of a wild type SPE-A toxin. Transformed cells are preferably microorganisms such as S. aureus, E. coli, or Salmonella species spp. Transformed microorganisms either include mutant SPE-A toxin or fragment thereof on their surface or are capable of secreting the mutant toxin. Transformed microorganisms can be administered as live, attenuated or heat killed vaccines.
The invention also includes methods of using vaccines and pharmaceutical compositions. Vaccines are administered to an animal in an amount effective to generate a protective immune response to at least one biological activity of a wild type SPE-A toxin. Preferably, the vaccine compositions are administered to humans and protect against the development of STSS. Pharmaceutical compositions are used in methods of stimulating T cell proliferation. The pharmaceutical compositions are especially useful in the treatment of cancers that are treated with interleukins, interferons or other immunomodulators, T cell lymphomas, ovarian and uterine cancers. A pharmaceutical composition is administered to a patient having cancer.
The mutant SPE-A toxins and/or fragments thereof and other vaccine compositions can be useful to generate a passive immune serum. Mutant SPE-A toxins or fragments thereof, DNA expression cassettes or vectors, or transformed microorganisms can be used to immunize an animal to produce neutralizing antibodies to at least one biological activity of wild type SPE-A. The neutralizing antibodies immunoreact with a mutant SPE-A toxin and/or fragment thereof and the wild type SPE-A toxin. Passive immune serum can be administered to an animal with symptoms of A streptococcal infection and STSS.